Vaccines have been very effective in protecting people from a wide variety of diseases, whether caused by viruses, bacteria, or fungi. The ability of vaccines to induce specific is protection against such a wide range of pathogenic organisms results from their ability to stimulate specific humoral antibody responses, as well as cell-mediated responses. This invention relates to a process for preparing such vaccines, and particularly to a process for making conjugates that are used in vaccines and immunogens. The invention further relates to the vaccines and immunogens produced from the conjugates made according to the invention, as well as to the use of these products.
It is often very desirable to induce immune responses against polysaccharides. For example, antibodies against a bacterial capsular polysaccharide can provide protection against that bacterium. Many polysaccharides, however, are poorly immunogenic, particularly in infants and young children. Furthermore, in both children and adults, there is usually no booster effect with repeated polysaccharide immunizations, and the principal antibody class is IgM. These features are all characteristic of so called "T cell independent" ("TI") antigens.
In many cases, the immunogenicity of polysaccharides can be enhanced by covalently linking proteins or T cell epitope-containing peptides or haptens to the polysaccharide. Certain other components, such as lipids, fatty acids, lipopolysaccharides, and lipoproteins, also are known to enhance the immunogenicity of the polysaccharide. As described in the "dual conjugate" patent application of Mond and Lees, conjugation of a protein to a polysaccharide can enhance the immune response to the protein as well as to the polysaceharide. See U.S. Pat. No. 5,585,100; U.S. patent application. Ser. No. 08/444,727 (filed May 19, 1995); and U.S. patent application Ser. No. 08/468,060 (filed Jun. 6, 1995). This patent and these patent applications each are entirely incorporated herein by reference. This effect also is described in A. Lees, et al., "Enhanced Inimunogenicity of Protein-Dextran Conjugates: I. Rapid Stimulation of Enhanced Antibody Responses to Poorly Immunogenic Molecules," Vaccine, Vol. 12, No. 13 (1994), pp. 1160-1166. This article is entirely incorporated herein by reference. In view of this potential for improving the immune response against polysaccharides, there is a need in the art for methods to covalently link proteins or other moieties to polysaecharides.
Ideally, the process of covalently linking proteins (or other moieties) to a polysaccharide should be done in a way to maintain antigenicity of both the polysaccharide and protein (or other) components and to minimize damage to necessary epitopes of each component. Furthermore, the linkage should be stable. Therefore, there is a need for a mild and gentle means for stably coupling proteins, peptides, haptens, or other moieties to polysaccharides.
Two main methods for coupling molecules together are used. In the first method, the means for coupling entails the crosslinking of a protein (or peptide or other moiety) directly to a polysaccharide (or some other moiety). Sometimes, however, a spacer molecule is needed between the coupled moieties, either to facilitate the chemical process and/or to enhance the immune response to the protein and/or the polysaccharide. In either method, it usually is necessary to activate or functionalize the polysaccharide before crosslinking occurs. Some methods of activating or functionalizing polysaccharides are described in W. E. Dick, et al., "Glycoconjugates of Bacterial Carbohydrate Antigens: A Survey and Consideration of Design and Preparation Factors," Conjugate Vaccines (Eds. Cruse, et al.), Karger, Basel, 1989, Vol. 10, pp. 48-114. This excerpt is entirely incorporated herein by reference. Additional activation methods are described in R. W. Ellis, et al. (Editors), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994), which book is entirely incorporated herein by reference.
One preferred method for activating polysaccharides is described in the "CDAP" (1-cyano-4-dimethylaminopyridine tetrafluoroborate) patent applications of Lees, U.S. patent application Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned); U.S. Pat. No. 5,651,971; U.S. Pat. No. 5,693,326; and U.S. patent application Ser. No. 08/482,666 (filed Jun. 7, 1995). These U.S. patents and patent applications each are entirely incorporated herein by reference. The use of CDAP also is described in Lees, et al., "Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate For Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents," Vaccine, Vol. 14, No. 3 (1996), pp. 190-198. This article also is entirely incorporated herein by reference.
Underivatized (or urnmodified) proteins can be conjugated to polysaccharides containing amine or hydrazide spacers using crosslinkers, such as gluteraldehyde or carboduimide. These methods, however, are prone to cause aggregation and homopolymerization, are difficult to control, and are likely to cause significant modification of the protein. These side effects are generally undesirable.
Instead of covalently linking proteins and polysaceharides via a spacer, a protein can be coupled to a polysaecharide with a spacer using heteroligation chemistry. In this procedure, the protein and polysaccharide components each are functionalized with chemical groups, and then the group(s) on the protein react selectively with the functional group(s) on the polysaccharide.
A common heteroligation method for linking proteins and polysaccharides via a spacer is through the use of a thio-ether linkage. See, for example, S. Marburg et al., Journal of the American Chemical Society, Vol. 108 (1986), beginning at page 5282, and U.S. Pat. No. 4,695,624, issued Sep. 22, 1987, which documents each are entirely incorporated herein by reference. Typically in this scheme, the polysaccharide is derivatized with an electrophilic group such as an .alpha.-haloacid, e.g., an iodoacetyl group, the protein is functionalized with thiol groups, and the two are combined. The thiol group attacks the .alpha.-carbon on the haloacid group and forms a thio-ether linkage.
Another known heteroligation method includes the formation of disulfides, as described in U.S. Pat. No. 5,204,098 to S. C. Szu et al., dated Apr. 20, 1993 (which patent is entirely incorporated herein by reference). In this method, the protein is derivatized with thiols and the polysaccharide with an exchangeable disulfide (e.g., a dithiopyridyl). Thiols on the protein undergo a disulfide exchange and form a conjugate with the polysaccharide.
A drawback to these heteroligation methods is the necessity for functionalizing the protein, which can involve multiple steps. The protein must be reacted with the chemical label, separated from the reaction products, and usually further concentrated. These steps can be costly and can result in the loss of protein material. Furthermore, unless care is taken, the protein thiols can oxidize, potentially causing homopolymerization and decreased conjugate yields. Additionally, not all the available functional groups on the protein participate in the crosslinking process, necessitating capping of these groups, thereby introducing additional, possibly deleterious epitopes. In the disulfide method described above, the S--S bond is susceptible to cleavage.
Furthermore, during heteroligation, it is known that .alpha.-haloacids are not perfectly selective for thiols and can react with other nucleophiles found in proteins, such as the side chains found on arginine, histidine, lysine, and methionine. The .alpha.-amine terminus, tyrosine, serine, glutamic acid, aspartic acid, and threonine can also react (see Wilchek et al., "Haloacetyl Derivatives," Methods in Enzymology, Vol. 46 (1977), beginning at page 153, which article is entirely incorporated herein by reference). Thus, in preparing conjugates where a thio-ether linkage is desired, care must be taken to avoid side reactions involving these other groups (see Hermanson, Bioconjugate Techniques, 1996 Academic Press, which book is entirely incorporated herein by reference).
For certain situations, however, these side reactions have proven useful. For example, haloacids have been found to be useful in the carboxymethylation of proteins (see F. R. N. Gurd, "Carboxymethylation," Methods of Enzymology, Vol. 11 (1967), pp. 532-541, which article is entirely incorporated herein by reference) and in affinity labeling of proteins (see Wilchek, et al., supra.). The use of iodoacetyl labeled peptides to cyclize peptides via properly positioned methionine, lysine, arginine, or histidines also has been described (see S. J. Wood, et al., "Novel Cyclization Chemistry Especially Suited for Biologically Derived, Unprotected Peptides," International Journal of Peptide and Protein Research, Vol. 39 (1992), pp. 533-539, which article is entirely incorporated herein by reference).
Many of these useful haloacyl reactions require that the haloacyl group be properly positioned in order for the reaction to be successful. Indeed, this is the basis of the affinity labeling described by Wilehek et al. Similarly, in Wood's process, it is essential that the iodoacetyl group be near the reactive amino acid for peptide cyclization to occur in good yield.
For the carboxymethylation process described by Gurd, high concentrations of the haloacyl reagent are required. Furthermore, the use of only a low molecular weight reagent is described (iodoacetic acid). Applicant has found that haloacyl functionalized polysaccharides are readily reacted with low molecular weight amines at room temperature and near neutral pH. In general, it is much more difficult to react high molecular weight macromolecules as compared to low molecular weight compounds.
Despite the various coupling and activation methods and reactions described in the various documents mentioned above, there is an on-going need in the art for improved methods for coupling biologically relevant molecules to one another to produce vaccines. This invention seeks to provide an improved coupling method for producing conjugates for vaccines and immunogens. As described in this application, a polyfunctional macromolecule, such as a protein, may react with haloacyl functionalized polysaccharides under reasonably mild conditions (e.g., moderate alkalinity) to effect a stable covalent linkage of the two molecules.